Dissection of zebrafish shha function using site-specific targeting with a Cre-dependent genetic switch

Abstract

Despite the extensive use of zebrafish as a model organism in developmental biology and regeneration research, genetic techniques enabling conditional analysis of gene function are limited. In this study, we generated Zwitch, a Cre-dependent invertible gene-trap cassette, enabling the establishment of conditional alleles in zebrafish by generating intronic insertions via in vivo homologous recombination. To demonstrate the utility of Zwitch, we generated a conditional sonic hedgehog a (shha) allele. Homozygous shha mutants developed normally; however, shha mutant embryos globally expressing Cre exhibited strong reductions in endogenous shha and shha target gene mRNA levels and developmental defects associated with null shha mutations. Analyzing a conditional shha mutant generated using an epicardium-specific inducible Cre driver revealed unique roles for epicardium-derived Shha in myocardial proliferation during heart development and regeneration. Zwitch will extend the utility of zebrafish in organ development and regeneration research and might be applicable to other model organisms.DOI: http://dx.doi.org/10.7554/eLife.24635.001

The role of endothelial cilia in postembryonic vascular development

“…41 Knock-in-based approaches to generating conditional mutant alleles appear even more promising than the conditional rescue approach we applied. 42,43 Although the rescue of ift54 mutant phenotype in this study was incomplete, most ift54 functions were restored by the conditional transgene used, and in contrast to ift54 tp49 homozygotes, which are extremely abnormal by 5 dpf and die shortly thereafter, 27,28 some transgenic ift54 tp49 homozygotes survive more than a year. The Zebrafish nose features non-motile cilia that project from multiciliated dendritic knobs of olfactory sensory neurons and motile cilia that differentiate from multiciliated cells at the rim of the olfactory pit.…”

“…Another Zebrafish study relied on a similar approach but used a bacterial artificial chromosome transgene and achieved a more complete conditional rescue . Knock‐in‐based approaches to generating conditional mutant alleles appear even more promising than the conditional rescue approach we applied …”

Zebrafish: Development of a Vertebrate Model Organism

“…In other cases, such as replacing neurons within the central nervous system, cells with stem-cell-like properties appear to be maintained throughout life (Than-Trong & Bally-Cuif, 2015; Wang et al, 2012). However, alternative inducible strategies such as estrogenreceptor controlled Cre recombinase or genetically encoded Cas9 would allow targeted gene disruption only in the cells of interest along with temporal control, such that gene functions in regeneration could be more specifically mapped (Sugimoto, Hui, Sheng, & Kikuchi, 2017). Teasing out the genetics of regenerative mechanisms has been somewhat more challenging than the development, in large part due to the significant overlap in pathways between development and regeneration.…”

A Regeneration Toolkit

“…The experimental advantages of zebrafish as a system include large clutches of 200 or more embryos, rapid external development, a reasonably short generation time of ~3 months, and a relatively small genome size that is half that of humans. Transgenesis, targeted knockout, and genome-editing based knock-ins are established, and protocols for cell-specific manipulations, and more recently knock-in and conditional alleles (Hoshijima et al, 2016; Sugimoto et al, 2017), are available though not yet commonplace. Non-saturating forward genetic screens for fin regeneration have been carried out in adult zebrafish (Johnson and Weston, 1995), identifying temperature-sensitive mutations in genes including fgf20a , laminin beta 1a , and mps1 , that bypass early development but block regeneration of amputated tail fins.…”

“…Zebrafish and axolotls have an expanding repertoire of Cre lines available to their researchers (Fei et al, 2017). loxP recognition sites for Cre have been inserted to flank coding sequences in zebrafish (Hoshijima et al, 2016), and an invertible gene trap cassette was recently used to knock out an Hh ligand gene in adult zebrafish to study heart regeneration (Sugimoto et al, 2017). Other methods for gene knockouts in adult animals could integrate additional spatiotemporal control for transgenic expression of Cas9 and gRNA delivery as in zebrafish embryos (Ablain et al, 2015; Yin et al, 2015), or electroporation of injected Cas9 protein and gRNA complexes in axolotls, as performed recently to target neural stem cells in spinal cord (Fei et al, 2016).…”